Motile processes in cells encompass a wide range of phenomena including muscle contraction and axoplasmic flow. On the molecular level most of these processes appear to be driven by the walking of myosin on actin filaments. We have recently developed a quantitative assay of the movement of myosin on actin which now makes it possible to study motility in vitro. In this assay myosin-coated fluorescent beads move unidirectionally along organized actin filament arrays isolated from the alga, Nitella. The velocity of the bead movement is determined by the myosin on the bead. This assay will be used to analyze myosin-dependent regulation of contraction (myosin isoenzyme substitution and light chain phosphorylation in smooth, cardiac and skeletal muscle myosins) in terms of effects on in vitro motility. To determine the effects of actin regulatory systems or different actin isozymes on motility an assay system will be developed using an in vitro polymerized actin substratum. Actin binding proteins such as filamin or spectrin can be analyzed for their effects on bead motility which will aid in understanding their roles in in vivo motile processes. Bead movement occurs in the absence of bipolar thick filaments because of direct attachment of myosin to the bead. Similar movements of cytoplasmic vesicles may occur by the attachment of myosin to them. Using the in vitro assay we will study axoplasmic flow squid axons to determine if vesicle movements are indeed driven by myosin attached to the vesicle. These studies will be expanded to determine how such vesicular movements are controlled and how many different vesicular movements are myosin driven. These studies can provide valuable insights into the molecular bases of cellular motility because we have for the first time an assay for measuring motility in vitro.